Sequence specific oligonucleotide probes (SSOP) can be used to identify nucleic acid sequences. Because the thermodynamic stability of matched duplexes is higher than a mismatched duplex, matched and mismatched duplexes can be identified by setting different hybridization temperatures and conditions.
Although hybridization techniques can be a useful tool for correctly identifying a complementary strand, current hybridization methods suffer from various limitations. For example, mismatch discrimination is affected by different mismatched base pairs, nucleic acid sequences, nucleic acid structures, etc. (See, Kawase et al., Nucleic Acids Res. 14(19):7727 (1986); Ikuta et al., Nucleic Acids Res. 15(2):797 (1987); Zhang et al., Biophysical J., 81(2):1133 (2001); Fang et al., Nucleic Acids Res. 29(15):3248 (2001)). The stability difference between a perfectly matched complement and a complement mismatched at only one base can also be quite small, corresponding to as little as 0.5° C. difference in their Tm value (Guo et al., Nat. Biotechnol. 15:331-5 (1997); and U.S. Pat. No. 5,780,233 to Guo et al.).
Some methods have been used to enhance the discrimination of single-base mismatches, such as the use of short probes having a length of no more than 15 nt. However, shorter probes are limited by their poor sequence specificity and low hybridization efficiency. In addition, the mismatch identification was also affected by GC percent, and the number and site of mismatched base. (See, Bhaumik et al., J Biomol Struct Dyn., 20(2):199 (2002); Letowski et al., J Microbiol Methods, 57(2):269 (2004)).
Alternatively, chemically modified nucleic acids such as locked nucleic acids (LNA) and peptide nucleic acids (PNA) may be used to enhance the discrimination of single-base mismatches. Locked nucleic acids (LNA) are oligonucleotide analogues containing a conformationally restricted nucleotide with a 2′-O, 4′-C-methylene bridge that induces thermal affinities when mixed with complementary single stranded DNA and RNA. (See, Petersen et al., Trends in Biotechnology, 21 (2): 74-81). Peptide nucleic acids (PNA) are synthetic chimeras of nucleobases linked to a peptide backbone. This spacing permits the bases to form, among other possible structures, standard base pairs with natural nucleic acids. However, the lack of the phosphodiester linkage, leading to an electronically neutral species, has important consequences for the base-pairing potential of PNA. Investigations of the stability and kinetics of PNA-DNA (and -RNA) duplex formation have confirmed and quantified the existence of strong base-pairing interactions under various conditions (See, Gabor, Proc. Natl. Acad. Sci. 95: 8562 (1998)).
In other methods, some base analogs such as 3-nitropyrrole, were inserted into oligonucleotide probes to increase the differences in thermal stability between hybrids formed with normal and single-nucleotide-variant DNA targets (See, Guo et al., Nat. Biotechnol., 15:331 (1997)).
However, existing methods used to enhance the discrimination of single-base mismatches are expensive because LNA, PNA and other base analogs are expensive to synthesize. Furthermore, optimal conditions such as the optimum number and site for these modified nucleic acids in an oligonucleotide probe are not known. Thus, there remains a need for improved low-cost methods for increasing hybridization specificity, and for enhancing the role of SSOP in the gene sequence analysis.